The molecular imaging of enzyme activity can be used to detect proteases and lipases in aggressive tumors, and can also be employed for reporter gene imaging by detecting activity of an expressed enzyme. We have developed a new type of contrast agent that detects enzyme activity with PhotoAcoustic Imaging (PAI).  Known as CAged MELANIN (CAMELANIN), the caging ligand of our agent is cleaved an enzyme to produce an unstable intermediate that spontaneously polymerizes into melanin. This melanin is detected with PAI. 

        We have synthesized CAMELANIN precursor in 3 steps with an 59.6% yield, and the caging ligand in 4 steps with a 59.6% yield.  We have combined the precursor and the caging ligand in 2 steps for a total 46.2% yield.  The modular design of our synthesis provides opportunities to develop additional agents with other caging ligands.  

        Before incubation with an enzyme, CAMELANIN is stable as a small molecule in water that does not absorb light.   After incubation with 1 unit of cathepsin B protease enzyme for 10 minutes, an insoluble, black pellet accumulates in the bottom of the tube, showing that an insoluble melanin-like polymer has been generated.  This change from a transparent sample to a sample with a black pellet is evidence for the detection of enzyme activity.

        Optical absorption at 540 nm was measured for natural eumelanin and for the melanin-like polymer (MLP) relative to the concentration of the monomer subunit in each polymer.  The MLP had 17% of the absorbance of eumelanin.  This result suggested that the MLP is less conjugated or more homogenously conjugated than eumelanin.  This result was expected, because the polymerization of the MLP occurs rapidly within 10 minutes. These results show that our product is melanin-like and still has excellent optical absorbance.

        PA spectra of eumelanin and the MLP were acquired in 1 nm increments.  The spectrum was normalized to the signal at 700 nm.  The normalized spectra showed that MLP has the same PA spectrum as eumelanin, demonstrating that MLP can be detected with MSOT.

        The rate of reaction was monitored using absorbance at 540 for 10 minutes using samples that had a range of monomer concentrations that were catalyzed by 1 unit of cathepsin B protease enzyme. The concentration-dependent reaction rate did not fit a first-order or second-order  model of chemical kinetics, or a model of Michaelis-Menten enzyme kinetics.  This result was expected, because the enzyme-catalyzed cleavage, spontaneous disassembly and spontaneous polymerization is a multistep reaction.  Importantly, the reaction rate is maximized at 2 mM monomer concentration, indicating a maximum for agent administration for rapid detection of enzyme activity.

        The reaction was monitored using absorbance at 540 nm for 10 minutes using 2 mM monomer and 1 unit of cathepsin B enzyme. In addition, 1 mM of enzyme inhibitor was added to a sample prior to incubation with enzyme.  A third sample was tested with no enzyme or inhibitor.  The sample treated with enzyme showed a strong increase in PA signal relative to the sample with no enzyme (p < 0.01).  The sample treated with the inhibitor before the enzyme largely eliminated the increase in PA signal.  This result further validated that the generation of MSOT signal from CAMELANIN is due to enzyme activity.

        Overall, these results demonstrate that CAMELANIN can detect enzyme activity and cn be used for photoacoustic imaging.

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Figure 1. Detection of enzyme activity with photoacoustic imaging. Before incubation with an enzyme, CAMELANIN is stable as a small molecule in water that does not absorb light.   After incubation with 1 unit of cathepsin B protease enzyme for 10 minutes, an insoluble, black pellet accumulates in the bottom of the tube, showing that an insoluble melanin-like polymer has been generated.  This change from a transparent sample to a sample with a black pellet is evidence for the detection of enzyme activity.

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